Use of a modified poxvirus for the rapid induction of immunity against a poxvirus or other infectious agents

ABSTRACT

The present invention relates to the rapid induction of a protective immune response against infectious agents using a poxvirus. An immune response can be induced by administering the poxvirus 7 to 2 days prior to infection with the infections agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of internationalapplication PCT/EP2006/001447, which was filed on Feb. 17, 2006, andclaimed the benefit of EP05003873.6, filed on Feb. 23, 2005, all ofwhich are incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the rapid induction of a protectiveimmune response against poxviruses and poxvirus infections such assmallpox by vaccination of an animal, including a human, with a poxvirusthat is replication incompetent in said animal, including the human. Anexample for such a poxvirus is a Modified Vaccinia virus Ankara (MVA).The invention further relates to the use of a recombinant poxvirus thatis replication incompetent in the animal, including the human, that isvaccinated with the virus, such as a recombinant MVA expressingheterologous antigens and/or antigenic epitopes for a rapid induction ofa protective immune responses against said heterologous antigen and/orantigenic epitope, e.g., against an antigen and/or antigenic epitopethat is part of an infectious agent.

BACKGROUND OF THE INVENTION

For many diseases, such as infectious diseases, vaccines have beendeveloped or are in the process of being developed. These vaccinesinduce a protective immune response within a certain time frame. Sincemost vaccines are used for the vaccination against diseases that arerather rare in the population, there is usually no need that thegeneration of the immune response be particularly rapid. However, thereare situations in which an immune response, such as a protective immuneresponse, should be generated as fast as possible. This may be the casein an outbreak of smallpox or in any other human poxvirus disease.

The causative agent of smallpox is the variola virus, a member of thegenus Orthopoxvirus. Vaccinia virus, also a member of the genusOrthopoxvirus in the family of Poxviridae, was used as a live vaccine toimmunize against smallpox. Successful worldwide vaccination withVaccinia virus culminated in the eradication of variola virus (Theglobal eradication of smallpox. Final report of the global commissionfor the certification of smallpox eradication; History of Public Health,No. 4, Geneva: World Health Organization, 1980). In the meantime, mostof the stocks of infectious variola viruses have been destroyed.However, it can not be excluded that poxviruses, inducing smallpox orsmallpox-like diseases, might again become a major health problem. Inaddition, there is a risk that a poxvirus disease of animals is spreadto humans.

Moreover, there may also be other situations in which it is desirable toinduce a rapid immune response. For example, it might be desirable toinduce a rapid immune response against diseases that are endemic in someparts of the world, if it is necessary to travel to such a country atshort notice.

BRIEF SUMMARY OF THE INVENTION

The invention encompasses the use of a poxvirus for the preparation of avaccine for the rapid induction of a protective immune response in ananimal, including a human, wherein the poxvirus is replicationincompetent in said animal, including in the human.

In one embodiment, the invention encompasses a method for the rapidinduction of a protective immune response in an animal, including ahuman, comprising the step of administering to the animal, including thehuman, a poxvirus that is replication incompetent in said animal,including in the human.

In one embodiment, the invention encompasses a use or method as above,wherein the protective immune response is generated within 7 days orless.

In one embodiment, the poxvirus is a Modified Vaccinia virus Ankara(MVA), particularly MVA 575, MVA 572 and, preferably, MVA-BN®.

The invention also encompasses uses or method as above, wherein thevirus is a cloned, purified virus. Particularly the inventionencompasses viruses obtained in a serum free cultivation process.

In one embodiment, the poxvirus is administered in a dose of 10⁵ to5×10⁸ TCID₅₀/ml. The poxvirus can be administered intravenously,intramuscularly or subcutaneously.

Preferably, the immune response is a protective immune response againsta poxvirus infection, preferably, a smallpox infection.

In one embodiment, the poxvirus is a recombinant poxvirus, preferably arecombinant MVA-BN. The poxvirus can comprise at least one heterologousnucleic acid sequence. Preferably, the heterologous nucleic acidsequence is a sequence coding for at least one antigen, antigenicepitope, and/or a therapeutic compound. The antigenic epitopes and/orthe antigens can be antigenic epitopes and/or antigens of an infectiousagent. The infectious agents can be a viruses, fungi, pathogenicunicellular eukaryotic or prokaryotic organisms, and parasiticorganisms. The viruses can be selected from the family of Influenzavirus, Flavivirus, Paramyxovirus, Hepatitis virus, Humanimmunodeficiency virus, or from viruses causing hemorrhagic fever. Theinfectious agent can be bacillus anthracis.

The invention includes a method for inducing a immune response againstan infectious agent in an animal comprising administering to the animalan immunogenic composition comprising an MVA, preferably MVA-BN, at 7 to2, 6 to 2, 5 to 2, 4 to 2, 3 to 2, or any other combination of thesedays (i.e., 6 to 4, 6 to 3, 5 to 4, 5 to 3, etc.) prior to infectionwith an infectious agent. In one embodiment, the infectious agent is areplication competent poxvirus. In a preferred embodiment, the animal isa human.

The invention further encompasses uses of the above methods and kitscomprising an immunogenic composition comprising an MVA, preferablyMVA-BN, and instructions to deliver the immunogenic composition at atime point between 7 and 2 days prior to exposure to an infectiousagent, including 7, 6, 5, 4, 3, or 2 days prior to exposure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Changes in body weight of differently vaccinated mice followingan intranasal challenge with 1× the MLD₅₀ of VV-WR. BALB/c mice werevaccinated subcutaneously with MVA-BN® or saline (PBS), or byscarification with Elstree, Dryvax®. Mice were treated with Saline (PBS)or Elstree, Dryvax 4 days, or with MVA-BN® 3 days or 2 days prior tochallenge with 4×10⁶ TCID₅₀/ml, 1×10⁷ TCID₅₀/ml, or 4×10⁷ TCID₅₀/mlVV-WR per mouse. Body weight was measured prior to challenge (day 0) andthen daily post challenge at the same time each day. The graphrepresents the average body weight changes per group over time usingnormalized (day 0 as baseline value) individual values.

FIG. 2. Changes in body weight of differently vaccinated mice followingan intranasal challenge with 12.5× the MLD₅₀ of VV-WR. BALB/c mice werevaccinated subcutaneously with MVA-BN® or saline (PBS), or byscarification with Elstree, Dryvax®. Mice were treated with Saline (PBS)or Elstree, Dryvax 4 days, or with MVA-BN® 3 days prior to challengewith 4×10⁶ TCID₅₀/ml, 1×10⁷ TCID₅₀/ml, or 4×10⁷ TCID₅₀/ml VV-WR permouse. Body weight was measured prior to challenge (day 0) and thendaily post challenge at the same time each day. The graph represents theaverage body weight changes per group over time using normalized (day 0as baseline value) individual values.

FIG. 3. Changes in body weight of differently vaccinated mice followingan intranasal challenge with 50× the MLD₅₀ of VV-WR. BALB/c mice werevaccinated subcutaneously with MVA-BN® or saline (PBS) or byscarification with Elstree, Dryvax®. Mice were treated with Saline (PBS)or Elstree, Dryvax 4 days, or with MVA-BN® 3 days prior to challengewith 4×10⁶ TCID₅₀/ml, 1×10⁷ TCID₅₀/ml, or 4×10⁷ TCID₅₀/ml VV-WR permouse. Body weight was measured prior to challenge (day 0) and thendaily post challenge at the same time each day. The graph represents theaverage body weight changes per group over time using normalized (day 0as baseline value) individual values.

FIG. 4. Titers of the VV within the lungs following challenge. BALB/cmice were vaccinated with a single administration of MVA-BN®,Elstree-BN, Dryvax, or treated with Saline (PBS). On days 2, 3 or 4 (asindicated in brackets) post-vaccination, mice were challenged witheither 1×, 12.5×, or 50×MLD₅₀ of VV-WR per mouse. The titers of VV-WR inthe lungs were determined by a standard plaque assay 4 to 8 days postchallenge and expressed at the man log₁₀ together with SEM.

FIG. 5. Comparison of antibody responses induced by MVA-BN®, Elstree-BNor Dryvax® immunizations. Balb/c mice were vaccinated with a singleadministration of MVA-BN®, Elstree-BN or Dryvax. Sera samples preparedon days 0 (pre-vaccination), 3, 4, 8, 12, 15 and 22 were analyzed byELISA for vaccinia-specific IgG titers. The titers have been plotted asGMT together with the SEM.

FIG. 6 A-D. Body weight loss and lung VV-WR titers in mice challengedwith either 1× (A & B) or 50× (C & D) MLD₅₀ VV-WR on days 7 or 14post-vaccination. Mice were vaccinated with either IMVAMUNE® (s.c.),Elstree-BN (scarification) or Dryvax® (scarification) and thenchallenged with either 1× or 50×MLD₅₀ VV-WR on day 7 or 14 postvaccination. Body weights were monitored and the animals sacrificed day5 post challenge and the titers of VV-WR in the lungs were determined bya standard plaque assay.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for the rapid induction of aprotective immune response in an animal, including a human, comprisingthe step of administering to the animal, including the human, a poxvirusthat is replication incompetent in said animal, including the human. Theinvention further relates to the use of said replication incompetentpoxvirus for the preparation of a vaccine for the rapid induction of aprotective immune response, as well as to a poxvirus as vaccine for therapid induction of a protective immune response, wherein the poxvirus isreplication incompetent in said animal, including the human.

The term “replication incompetent poxvirus” and the synonymous termvirus that is “not capable of being replicated to infectious progenyvirus” both refer to poxviruses that do not replicate at all in thecells of the vaccinated animal, and to viruses that show a minorresidual replication activity that is controlled by the immune system ofthe animal, including the human, to which the poxvirus is administered.

According to an embodiment of the present invention, the replicationincompetent poxviruses are viruses that are capable of infecting cellsof the animal, including the human, in which the virus is used asvaccine. Viruses that are “capable of infecting cells” are viruses thatare capable of interacting with the host cells to such an extent thatthe virus, or at least the viral genome, becomes incorporated into thehost cell. Although the viruses used according to the present inventionare capable of infecting cells of the vaccinated animal, including ahuman, they are either not capable of being replicated to infectiousprogeny virus in the cells of the vaccinated animal, or they show only aminor residual replication activity that is controlled by the immunesystem of the animal, including the human, to which the poxvirus isadministered.

It is to be understood that, a virus that is capable of infecting cellsof a first animal species but not capable of being replicated toinfectious progeny virus in said cells may behave differently in asecond animal species. For example, for humans, MVA-BN® and itsderivatives (see below) are viruses that are capable of infecting cellsof the human but that are not capable of being replicated to infectiousprogeny virus in human cells. The same viruses are very efficientlyreplicated in chickens; i.e. in chickens, MVA-BN® is a virus that iscapable of infecting cells and capable of being replicated to infectiousprogeny virus. It is known to the person skilled in the art which virushas to be chosen for a specific animal species. A test that allowsdetermining whether a virus is capable or not capable of beingreplicated in severely immunocompromised mice is disclosed in WO02/42480 and uses the AGR129 mice strain (see below), or any other mousestrain that is incapable of producing mature B and T cells and as suchis severely immune compromised and highly susceptible to a replicatingvirus. The results obtained in this mouse model are indicative forhumans.

Some MVAs, such as MVA-572 and MVA-575, are not as attenuated as MVA-BN,as elucidated in WO 02/42480. For example, MVA-572 is capable of killingseverely immunocompromised mice.

According to an embodiment of the present invention, the virusesaccording to the present invention are capable of being replicated in atleast one type of cells of at least one animal species. Thus, it ispossible to amplify the virus prior to administration to the animal thatis to be vaccinated and/or treated. By way of example, reference is madeto MVA-BN® that can be amplified in CEF (chicken embryo fibroblasts)cells but that is a virus that is not capable of being replicated toinfectious progeny virus in humans.

According to an embodiment of the present invention, Modified Vacciniavirus Ankara (MVA) is used in humans and several animal species, such asmice and non-human primates. MVA is known to be exceptionally safe. MVAhas been generated by long-term serial passages of the Ankara strain ofVaccinia virus (CVA) on chicken embryo fibroblasts (for review see Mayr,A., Hochstein-Mintzel, V. and Stickl, H. [1975] Infection 3, 6-14; SwissPatent No. 568, 392). Examples for MVA virus strains that have beendeposited in compliance with the requirements of the Budapest Treaty andthat are useful in the practice of the present invention are strains MVA572 deposited at the European Collection of Animal Cell Cultures(ECACC), Salisbury (UK) with the deposition number ECACC 94012707 onJan. 27, 1994, MVA 575 deposited under ECACC 00120707 on Dec. 7, 2000,and MVA-BN® deposited with the number 00083008 at the ECACC on Aug. 30,2000. Although MVA-BN is preferred to its higher safety (lessreplication competent), all MVAs are suitable for this invention.

According to an embodiment of the present invention, the MVA strain isMVA-BN® and its derivatives. A definition of MVA-BN® and its derivativesis given in PCT/EP01/13628.

In short, MVA-BN® and its derivatives as disclosed in PCT/EP01/13628 arecharacterized in having at least one, at least two, at least three orall of the following properties:

-   -   (i) capability of reproductive replication in chicken embryo        fibroblasts (CEF) and in the cell line BHK, but no capability of        reproductive replication in human cell lines. According to an        embodiment of the present invention the human cell lines are the        human bone osteosarcoma cell line 143B, the human keratinocyte        cell line HaCat and the human cervix adenocarcinoma cell line        HeLa,    -   (ii) failure to replicate in vivo in severely immune compromised        mice,    -   (iii) induction of a higher immunogenicity compared to the known        strain MVA 575 (ECACC V00120707) in a lethal challenge model        and/or    -   (iv) induction of at least substantially the same level of        immunity in vaccinia virus prime/vaccinia virus boost regimes        when compared to DNA-prime/vaccinia virus boost regimes.

For detailed information regarding the assays used to determine whethera MVA strain has one or more of the above features (i) to (iv) referenceis made to WO 02/42480 (PCT/EP01/13628). This publication also discloseshow viruses having the desired properties can be obtained. In thefollowing it is shortly summarized how the person skilled in the art cantest whether an MVA strain has one or more of said features and is,thus, a virus according to said embodiment of the present invention. Thefollowing summary is not to be understood as to limit the relevance ofWO 02/42480 for the present application to the following information.Instead, WO 02/42480 is herewith incorporated in its entirety byreference.

The term “not capable of reproductive replication” in human cell linessuch as the cell lines HaCAT (Boukamp et al. 1988, J Cell Biol 106(3):761-71) or HeLa is used in the present application as defined in WO02/42480. Thus, a virus that is “not capable of reproductivereplication” in a cell line is a virus that shows an amplification ratioof less than 1 in said cell line. The “amplification ratio” of a virusis the ratio of virus produced from an infected cell (Output) to theamount originally used to infect the cells in the first place (Input). Aratio of “1” between Output and Input defines an amplification statuswherein the amount of virus produced from the infected cells is the sameas the amount initially used to infect the cells. According to anembodiment of the present invention the viruses that are “not capable ofreproductive replication” in human cell lines may have an amplificationratio of 1.0 (average value) or less, or even 0.8 (average value) orless, in any of the above human cell lines HeLa, HaCat and 143B.

The term “average” as used in the present application refers to theaverage values obtained from at least 2, but possibly 3, 4, 5, 6, 7, 8,9, 10 or more experiments. It will be understood by a person skilled inthe art that single experiments may deviate from average values due tothe inherent variability of biological systems.

The term “failure to replicate in vivo” is used in the presentapplication as defined in WO 02/42480. Thus, said term refers to virusesthat do not replicate in the mouse model as explained in WO 02/42480.The mice used in WO 02/42480 are incapable of producing mature B- andT-cells (AGR 129 mice). MVA-BN® and its derivatives do not kill AGR129mice within an average time period of at least 45 days (average value),such as within at least 60 days (average value), or within 90 days(average value) after the infection of the mice with 10⁷ pfu virusadministered intraperitonealy. According to an embodiment of the presentinvention, the viruses that show “failure to replicate in vivo” arefurther characterized in that no virus can be recovered from organs ortissues of the AGR129 mice 45 days (average value), alternatively 60days (average value), and alternatively 90 days (average value), afterthe infection of the mice with 10⁷ pfu virus administered intraperitonealy. Instead of the AGR129 mice, any other mouse strain can beused that is incapable of producing mature B and T cells, and as such isseverely immune compromised and highly susceptible to a replicatingvirus. The data obtained in said mouse model are predictive for humans.Thus, according to an embodiment, the viruses of the present invention,such as MVA-BN® and its derivatives, do not replicate at all in humans.In applying the definition in the section related to the terms“replication incompetent poxvirus” and “virus that is not capable ofbeing replicated to infectious progeny virus” to the replicationbehavior of MVA-BN® and its derivatives in humans, additional virusesthat are within the scope of the present invention are those that show aminor residual replication activity that is controlled by the immunesystem of the human to which the poxvirus is administered.

The details of the lethal challenge experiment used to determine whetheran MVA strain has “a higher immunogenicity compared to the known strainMVA 575” are explained in WO 02/42480. In such a lethal challenge model,unvaccinated mice die after the infection with replication competentvaccinia strains, such as the Western Reserve strain L929 TK+ and IHD-J.The infection with replication competent vaccinia viruses is referred toas “challenge” in the context of description of the lethal challengemodel. Four days after the challenge, the mice are usually killed andthe viral titer in the ovaries is determined by standard plaque assaysusing VERO cells. The viral titer is determined for unvaccinated miceand for mice vaccinated with MVA-BN® and its derivatives. Morespecifically MVA-BN® and its derivatives are characterized in that, inthis test, after the vaccination with 10² TCID₅₀/ml virus, the ovaryvirus titers are reduced by at least 70% (average value), alternativelyby at least 80% (average value), alternatively by at least 90% (averagevalue), compared to unvaccinated mice.

A vaccinia virus, such as an MVA strain, is regarded as inducing atleast substantially the same level of immunity in vaccinia virusprime/vaccinia virus boost regimes when compared to DNA-prime/vacciniavirus boost regimes if the CTL response as measured in one of the “assay1” and “assay 2” as disclosed in WO 02/42480 is at least substantiallythe same in vaccinia virus prime/vaccinia virus boost regimes whencompared to DNA-prime/vaccinia virus boost regimes. According to anembodiment of the present invention, the CTL response is at leastsubstantially the same in vaccinia virus prime/vaccinia virus boostregimes when compared to DNA-prime/vaccinia virus boost regimes asmeasured in both of the “assay 1” and “assay 2” as disclosed in WO02/42480. According to an embodiment of the present invention, the CTLresponse after vaccinia virus prime/vaccinia virus boost administrationis higher in at least one of the assays, when compared toDNA-prime/vaccinia virus boost regimes. According to an embodiment ofthe present invention the CTL response is higher in both assays.

According to an embodiment of the present invention, the derivatives ofMVA-BN® are characterized (i) in being capable of reproductivereplication in chicken embryo fibroblasts (CEF) and in the Baby hamsterkidney cell line BHK, but not capable of reproductive replication inhuman cell lines, wherein according to an embodiment of the presentinvention the human cell lines are the human bone osteosarcoma cell line143B, the human keratinocyte cell line HaCat and the human cervixadenocarcinoma cell line HeLa; and (ii) by a failure to replicate invivo in severely immune compromised mice.

According to an embodiment of the present invention, the virus is acloned purified virus, such as a monoclonal virus.

According to an embodiment of the present invention, the virus is avirus that has been produced/passaged under serum free conditions toreduce the risk of infections with agents contained in serum.

MVA according to the present invention is administered in aconcentration range of 10⁴ to 10⁹ TCID50/ml, e.g. in a concentrationrange of e.g. 10⁵ to 5×10⁸ TCID₅₀/ml or in a concentration range of e.g.10⁶ to 10⁸ TCID₅₀/ml. The actual concentration depends on the type ofthe virus and the animal species to be vaccinated. For MVA-BN® a typicalvaccination dose for humans comprises 5×10⁷ TCID₅₀ to 5×10⁸ TCID₅₀, e.g.about 1×10⁸ TCID₅₀, administered subcutaneously.

According to an embodiment of the present invention, the poxvirus asdefined above, e.g. an MVA strain, such as MVA-BN® and its derivativesis administered in a single administration to induce a rapid protectiveimmune response. Clinical data have shown that a single vaccination withMVA-BN® resulted in a detectable immune response in almost 100% of thevaccinated individuals.

According to another embodiment of the present invention the poxvirus asdefined above, e.g. an MVA strain, such as MVA-BN® and its derivativesmay also be used in homologous prime boost regimes. In other words, itis possible to use a poxvirus such as MVA for a first vaccination and toboost the immune response generated in the first vaccination byadministration of the same or a related strain of the poxvirus than theone used in the first vaccination. The poxvirus as defined above, e.g.an MVA strain, such as MVA-BN® and its derivatives may also be used inheterologous prime-boost regimes in which one or more of thevaccinations is done with a poxvirus as defined above and in with one ormore of the vaccinations is done with another type of vaccine, e.g.another virus vaccine, a protein or a nucleic acid vaccine.

The mode of administration may be intravenously, intradermal,intranasal, or subcutaneously. Any other mode of administration may beused.

The poxvirus used according to the present invention may be anon-recombinant poxvirus such as an MVA strain, e.g. MVA-BN® and itsderivatives. In this case, the vaccination may be done to rapidly inducea protective immune response against a poxvirus infection such assmallpox.

Thus, according to the present invention, the poxvirus as defined above,such as an MVA strain, e.g. MVA-BN® and its derivatives is suitable torapidly induce a protective immune response against smallpox. This isexemplified in Example 1, where it is compared how long it takes until aprotective immune response is generated in mice against a pathogenicvaccinia virus strain, after vaccination with MVA-BN® (a strainaccording to the present invention) and with non-MVA strains such asDryvax® and Elstree. These non-MVA strains are fullyreplication-competent, in contrast to MVA-BN®. It is shown that MVA-BN®clearly has improved properties compared to Elstee and Dyvax, in that asingle vaccination of mice with MVA-BN® leads to a significantprotective immune response, when the vaccination is administered withinfour, three and even two days before exposure to the pathogenic vacciniavirus strain. For example, this is demonstrated by assessing, in themice's lungs, the titer of a pathogenic Vaccinia virus strain Westernreserve (VV-WR) administered to a mouse two, three or four days afterthe vaccination with MVA-BN®. When the mice were challenged with12.5×MLD₅₀ of VV-WR three days after the vaccination, no VV-WR viraltiter could be detected in mice vaccinated with a standard dose ofMVA-BN®, whereas the mice vaccinated with Dryvax® or Elstree were notprotected and had a lung titer that was very similar to the titer ofunvaccinated control mice. When the mice were challenged with 50×MLD₅₀four days after the vaccination, no VV-WR viral titer could be detectedin mice vaccinated with a standard dose of MVA-BN®, whereas the micevaccinated with Dryvax® or Elstree were not protected and had a lungtiter that was very similar to the titer of unvaccinated control mice.The term MLD₅₀ refers to the concentration of a pathogenic Vacciniavirus strain at which 50% of the inoculated mice die.

It is to be noted that the mice data are predictive for humans.Moreover, it is to be taken into account that concentrations of apathogenic virus that are 50 times the lethal dose usually do not occurin nature, in particular not for human poxviruses that induce smallpox.

According to an embodiment of the present invention, the term “rapidinduction of a protective immune response in an animal, including ahuman” refers preferably to the generation of a protective immuneresponse within 7 days or less, 6 days or less, 5 days or less, 4 daysor less, 3 days or less, or even 2 days or less, after the vaccinationwith a virus according to the present invention. This is unexpectedsince it was a dogma in the state of the art that it takes at least 10to 14 days until a protective immune response is generated againsttraditional smallpox vaccines, based on replicating vaccinia virusstrains. The rapidity of the induction of a protective immune responsecan be evaluated in the animal model described in the examples section.Said model is also predictive for humans. Thus, according to anembodiment of the present invention a poxvirus vaccine is effective ininducing a rapid immune response in mice if after vaccination of micewith an effective dose of a poxvirus vaccine such as MVA, e.g. MVA-BN®and derivatives thereof, and challenge with 1×, 12.5×, and 50×MLD₅₀ ofVV-WR four days after vaccination, the lung titers of the virus arebelow an average of 5×10³ pfu (corresponding to log 3.69), as determinedin the test system described in the examples section. Alternatively,according to an embodiment of the present invention, a poxvirus vaccineis effective in inducing a rapid immune response if the lung titervalues are below an average of 5×10³ pfu (corresponding to log 3.69)after a challenge with 1×, and 12.5×MLD₅₀ of VV-WR three days aftervaccination with an effective dose of the poxvirus vaccine.

In a broader sense, a virus leads to a rapid induction of a protectiveimmune response in mice if said virus behaves similarly to MVA-BN® inthe lung titer assay and the body weight assay described in the examplessection. Thus, the limits, threshold values, conditions and parametersas described in the examples section, also apply in a general sense forother poxvirus vaccines that are regarded as rapid inducers of aprotective immune response. From this, it is obvious that the data andinformation given in the examples section can be generally used tosupplement any missing data and information in this paragraph, such asinformation relating to the description of the test system.

Alternatively, the rapidity of the induction of the protective immuneresponse can be evaluated with the serum conversion test explainedbelow; in this context, the time point at which seroconversion isobserved is regarded as the time point at which the protective immuneresponse was induced.

According to an embodiment of the present invention the animal,including a human, is an animal that is naäve with respect to poxvirusinfections, i.e. an animal that has never been in contact withpoxviruses and that has not been vaccinated with poxvirus vaccines.

According to a related embodiment the animal, including a human, is ananimal that was in contact with poxviruses and/or that was vaccinatedwith a poxvirus vaccine. Such animal, including a human, might haveraised an immune response against poxviruses and/or poxvirus vaccines,such as MVA.

The term “protective immune response” means that the vaccinated animalis able to control an infection with the pathogenic agent against whichthe vaccination was done. Usually, the animal having developed a“protective immune response” develops only mild to moderate clinicalsymptoms or no symptoms at all. Usually, an animal having a “protectiveimmune response” against a certain agent will not die as a result of theinfection with said agent.

As pointed out above, a concentration of MVA-BN® or a derivative thereofused for the generation of a protective immune response in humansagainst smallpox is in the range of 5×10⁷ TCID₅₀ to 5×10⁸ TCID₅₀, suchas 1×10⁸ TCID₅₀, wherein the virus may be administered subcutaneously orintramuscularly.

It seems as if the mechanism of the development of a rapid immuneprotection after vaccination with a poxvirus as defined above, such asan MVA strain, e.g. MVA-BN® and its derivatives, depends on whether thevaccinated animal, including a human, is a naäve animal (that was neverin contact with a poxvirus before) or an animal that had been in contactwith a poxvirus before (e.g. by vaccination). In the naäve animal,including a human, the administration of the poxvirus according to thepresent invention, such as MVA-BN® or its derivatives efficiently primesthe immune system, even if neutralizing antibodies may not be detectablein the first few days after vaccination (see Example 2). The infectionwith a pathogenic virus boosts the immune system, in such way that theeffectively primed immune system can control said infection unexpectedlyeffective and fast (see Example 2). Thus, naäve animals that have beenvaccinated with a virus according to the present invention are readilyprotected against the infection with the pathogenic virus against whichthe vaccination is done after a single vaccination only.

The viruses as defined according to the present invention, such asMVA-BN® and its derivatives also are unexpectedly efficient and fast inboosting the earlier vaccination in animals that have been in contactwith a poxvirus before, so that a protective immune response is alsorapidly generated in this situation.

The rapidity of the induction of a protective immune response is alsoreflected by an unexpectedly fast seroconversion after vaccination ofanimals, including humans, with a virus according to the presentinvention such as MVA, e.g. MVA-BN® and its derivatives. In non-humanprimates, it is shown that seroconversion occurs within less than 10days, e.g. within 7 days, which is one week faster than theseroconversion after vaccination with other smallpox vaccines, such asElstree. In the following it is described how the seroconversion aftervaccination with MVA-BN® and its derivatives is evaluated. The same testprinciple is applied mutatis mutandis if the seroconversion aftervaccination with other viruses is tested. The only modification that isrequired to assess the seroconversion induced by said other viruses isto quantify the total IgG antibodies specific for said other viruses,instead of quantifying the total MVA-BN® specific IgG antibodies. Thecut off values and the criteria to evaluate whether a sample is positiveare determined in basically the same way, with optional minormodifications that are within the skills of the skilled artisan. Toassess for a seroconversion after vaccination with MVA-BN® (or itsderivatives), total MVA-BN® specific IgG antibodies are quantified intest sera using a direct Enzyme-Linked Immunosorbent Assay (ELISA). Adetailed description of a study applying this method is provided inExample 2.

The ELISA is a sensitive method used for the detection of antibodies insera. The MVA-BN® specific ELISA is a standard binding ELISA used todetect total IgG antibodies in human test sera. ELISA results areexpressed as an end point antibody titer obtained by directdetermination of logarithmic trend lines. A cut off, or end pointabsorbance of 0.35 has been defined. The end point titer of the sampleis determined by generating a logarithmic plot, e.g. by using thecommercially available computer program Excel (expressing opticaldensity (OD) on the y axis and the log of the sera dilution on the xaxis). Again, the data in non-human primates are predictive for humans.A test sample is deemed positive when the OD of the sample is greaterthan 0.35 at a 1:50 dilution of a test sample. The geometric mean titer(GMT) is calculated by taking the antilogarithm of the mean of the log10 titer transformations. The GMT is usually the reported titer forELISA titers.

Seroconversion rate is defined as percentage of initially seronegativesubjects with appearance of antibody titers ≧1:50 in the MVA-specificIgG ELISA. Thus, according to an embodiment of the present invention theterm “rapid induction of a protective immune response in an animal,including a human” refers to a seroconversion as defined above, with thetest as defined above, within 10 days or less, 7 days or less, 6 days orless, 5 days or less, 4 days or less, 3 days or less, or even 2 days orless, after the vaccination with a virus according to the presentinvention.

The poxvirus as defined above such as an MVA strain, e.g. MVA-BN® andits derivatives may also be a recombinant poxvirus strain such asrecombinant MVA-BN® or its derivatives. The recombinant virusesaccording to the present invention, such as recombinant MVA-BN® and itsderivatives, may contain at least one heterologous nucleic acidsequence. The term “heterologous” is used hereinafter for anycombination of nucleic acid sequences that is not normally foundintimately associated with the virus in nature. The heterologoussequences may be antigenic epitopes or antigens, which are selected fromany non-vaccinia source. According to an embodiment of the presentinvention, said recombinant virus expresses one or more antigenicepitopes or antigens, which are antigenic epitopes or antigens from aninfectious agent. The infectious agent may be any infectious agent, suchas a virus, a fungus, a pathogenic unicellular eukaryotic or prokaryoticorganism, and a parasitic organism. Examples of infectious agents arePlasmodium falciparum, Mycobacteria, Influenza virus, Flaviviruses,Paramyxoviruses, Hepatitis viruses, Human immunodeficiency viruses,viruses causing hemorrhagic fever such as Hantaviruses or Filoviruses,i.e., Ebola and Marburg virus. The infectious agent can be bacillusanthracis, which causes anthrax.

According to this embodiment the recombinant poxvirus as defined abovemay not only be used to induce a rapid immune response against apoxvirus infection, but may also (or alternatively) be used to induce arapid immune response against the heterologous antigenic epitope/antigenexpressed from the heterologous nucleic acid comprised in therecombinant virus. Thus, by way of example, if a recombinant MVAexpresses an HIV epitope or a yellow fever virus epitope, therecombinant MVA may be used to induce a rapid immune response againstHIV or Yellow fever virus, respectively.

It is also within the scope of the present invention that therecombinant virus may alternatively express an antigenic epitope/antigenthat further increases the immunogenicity of MVA.

The recombinant virus used according to the present invention may alsocomprise a heterologous gene/nucleic acid expressing a therapeuticcompound. A “therapeutic compound” encoded by the heterologous nucleicacid in the virus can be, for example, a therapeutic nucleic acid suchas an antisense nucleic acid, or a peptide, or a protein with desiredbiological activity.

According to an embodiment of the present invention, the expression ofheterologous nucleic acid sequence may be under the transcriptionalcontrol of a poxvirus promoter. An example of a suitable poxviruspromoter is the cowpox ATI promoter (see WO 03/097844).

According to an embodiment of the present invention, the insertion of aheterologous nucleic acid sequence is done into a non-essential regionof the virus genome. According to another embodiment of the invention,the heterologous nucleic acid sequence is inserted at a naturallyoccurring deletion site of the MVA genome (disclosed in PCT/EP96/02926).According to a further alternative, the heterologous sequence may beinserted into an intergenic region of the poxviral genome (see WO03/097845). Methods on how to insert heterologous sequences into thepoxviral genome are known to a person skilled in the art.

The invention also encompasses kits for the induction of a protectiveimmune response. In one embodiment, the kit comprises an immunogeniccomposition comprising an MVA and instructions for the delivery of theimmunogenic composition. The MVA is preferably MVA-BN. Preferably, theimmunogenic composition contains 10⁵ to 5×10⁸ TCID₅₀/ml of MVA. Theinstructions for delivery of the immunogenic composition can direct thedelivery at various time points prior to exposure to an infectiousagent. These time points can include time points between 7 and 2 daysprior to exposure to an infectious agent. In this context, an “exposure”means contact with the infectious agent itself, or with an animal(human) harboring the infectious agent. The time points can also includetime points between 6 and 2 days, 5 and 2 days, 4 and 2 days, 3 and 2days, and 2 days prior to exposure to an infectious agent. For example,the instructions can direct that the immunogenic composition can bedelivered at 7, 6, 5, 4, 3, or 2 days prior to exposure to an infectiousagent. Preferably, the infectious agent is smallpox or bacillusanthracis. The instructions can direct that the MVA be administered MVAintravenously, intramuscularly, and/or subcutaneously.

All definitions given above for the embodiment regarding non-recombinantviruses apply also for the embodiment concerning recombinant viruses.

The specification is most thoroughly understood in light of the citedreferences, all of which are hereby incorporated by reference in theirentireties.

EXAMPLES

The following examples will further illustrate the present invention. Itwill be well understood by a person skilled in the art that the providedexamples in no way may be interpreted in a way that limits theapplicability of the technology provided by the present invention tothis examples.

Example 1 Onset of Protection in MVA-BN®, Elstree, or Dryvax® VaccinatedMice Challenged with 1×, 12.5×, or 50× the MLD₅₀ of Vaccinia VirusVV-Western Reserve (VV-WR) on Days 2 Through 4 Post-Vaccination:Assessment by Body Weight and Lung Titers 1. INTRODUCTION

A murine intranasal vaccinia challenge model has been developed to testthe efficacy of smallpox vaccine candidates. In this model, mice arevaccinated with vaccines the efficiency of which is to be determined.Control mice receive a saline control instead of the vaccine. Severaldays after the vaccination, the mice are infected with a pathogenicVaccinia virus strain, such as the vaccinia virus strain Western Reserve(VV-WR). The murine lethal dose 50 (MLD₅₀) of the vaccinia virus strainWestern Reserve (VV-WR) was determined to be 3.6×10⁴ TCID₅₀ inunvaccinated mice.

In a preliminary study it was shown that MVA-BN®-vaccinated BALB/c micechallenged with either 25× or 50× the MLD₅₀ of VV-WR, quickly recoveredfrom the viral challenge, showed no clear signs of clinical symptoms,and no pathological lesions were present in the lungs of these animals.In another preliminary study, the time required after vaccination toestablish protection from a lethal challenge with VV-WR wasinvestigated: challenging of MVA-BN®-vaccinated mice with a sub-lethaldose of VV-WR 3 days after the vaccination, revealed protection (withregard to body weight loss and viral lung titers). The objective of thisexample was to narrow down the time required after MVA-BN® (or Elstreeor Dryvax®) vaccination to obtain protection following a lethalchallenge with VV-WR.

2. VIRUSES AND CONTROLS

Test vaccine 1

Modified Vaccinia Ankara-Bavarian Nordic (MVA-BN®/IMVAMUNE®), in aconcentration of 5.0E+08 TCID₅₀/ml. Formulation: in 10 mM Tris 140 mMNaCl pH7.4.

No further dilutions of the MVA-BN® stock were made in the MVA-BN®vaccinated groups and 200 μl was administered subcutaneously, resultingin a final dose of 1.0E+08 TCID₅₀.

Test Vaccine 2

Vaccinia virus strain Elstree with a nominal concentration of 1.0E+08TCID₅₀/ml. Formulation: in 10 mM Tris 140 mM NaCl pH7.4.

No further dilutions of the Elstree stock were made and 2.5 μl wasadministered via scarification on the tail of each mouse, resulting in afinal dose of 2.5E+05 TCID₅₀.

Test vaccine 3

Dryvax® with a nominal concentration of 2.9E+07 TCID₅₀/ml. Formulation:in 10 mM Tris 140 mM NaCl pH7.4.

No further dilutions of the Dryvax® stock were made and 8 μl wasadministered via scarification on the tail of each mouse, resulting in afinal dose of 2.5E+05 TCID₅₀.

Each of the test vaccines 1 to 3 was administered with its optimal doseand route of administration.

Challenge Virus

Vaccinia Virus Western Reserve (VV-WR) with a nominal concentration of4.0E+08 TCID₅₀/ml.

The following dilutions of VV-WR (4.0E+08 TCID₅₀/ml) were made togenerate a final working stock of 8.0E+05 TCID₅₀/ml and 4.0E+07TCID₅₀/ml:

For 1× the MLD₅₀ of VV-WR/mouse (8.0E+05 TCID₅₀/ml working stocksuspension): 100 μl of the VV-WR stock 4.0E+08 TCID₅₀/ml was added to900 μl of PBS, mixed by vortexing (to give a concentration of 4.0E+07TCID₅₀/ml); 100 μl of this suspension was transferred to 900 μl of PBS,again mixed by vortexing (to give a concentration of 4.0E+06 TCID₅₀/ml);600 μl of this suspension was added to 2400 μl of PBS, mixed byvortexing to give a final concentration of 8.0E+05 TCID₅₀/ml.For 12.5× the MLD₅₀ of VV-WR/mouse (1×10⁷ TCID50/ml working stocksuspension): 75 μl of the VV-WR stock 4×10⁸ TCID50/ml was added to 2925μl of PBS and mixed by vortexing to give a final concentration of 1×10⁷TCID50/ml.For 50× the MLD₅₀ of VV-WR/mouse (4.0E+07 TCID₅₀/ml working stocksuspension): 300 μl of the VV-WR stock 4.0E+08 TCID₅₀/ml was added to2700 μl of PBS and mixed by vortexing to give a final concentration of4.0E+07 TCID₅₀/ml.

Saline Control:

In the Saline Control groups, 200 μl PBS (as supplied by themanufacturer) was used for injecting individual mice subcutaneously

3. METHODS AND EXPERIMENTAL DESIGN Test System

Specific Pathogen Free (SPF) female Balb/c mice H-2d were obtained fromTaconic M&B, P.O. Box 1079, DK-8680 Ry, Denmark. Number of animals inthe study: 60. Age at initiation of challenge: 9 weeks. Body weightrange at initiation of challenge: 18-23 grams. The BALB/c mouse strainhas been used extensively to test the immunogenicity and efficacy ofsmallpox vaccines. The strain is highly susceptible to VV-WR challenge.

The experiments were carried out in accordance with theDyreforsøgstilsynet of Denmark

Allocation to treatment groups: On arrival, animals were randomlyallocated to a treatment group consisting of 5 animals per test group(and cage).

Justification of the dose level: MVA-BN® was used at an optimal dosethat has been demonstrated in previous experiments to induce stronghumoral and cell mediated immune responses in mice. Elstree and Dryvax®were used at a dose suggested for humans.

Vaccination and Challenge Schedule

A total of 80 mice were used in this study (see Table 1 below). Micewere either challenged with 1×, 12.5×, or 50× the MLD₅₀ of VV-WR, 4 daysafter vaccination with either MVA-BN®, or Elstree, or Dryvax®. Inadditional groups, mice have been challenged with 1×, 12.5×, or 50× theMLD₅₀ of VV-WR, 3 days after vaccination with MVA-BN®; or mice have beenchallenged with 1× the MLD₅₀ of VV-WR, 2 days after vaccination withMVA-BN®. In control groups, mice have been challenged with 1×, 12.5×, or50× the MLD₅₀ of VV-WR, without prior vaccination. Control animalsreceiving the 1× and 12.5×, or 50× the MLD₅₀ of VV-WR were sacrificed 5or 4 days after challenge, respectively, in case body weight lossexceeded 20% from initial body weight, or in case the animals sufferedfrom dyspnea. This was done to reduce suffering.

TABLE 1 Dose and challenge regimen of mice used to investigate the onsetof MVA-BN ® protection Challenge Time between Dose vaccination Sample (xthe MLD₅₀ and Group Vaccination Size of VV-WR) challenge 1 Saline 5 1× 4days 2 Saline 5 12.5×   4 days 3 Saline 5 50×  4 days 4 MVA-BN ® 5 l× 4days 5 MVA-BN ® 5 12.5×   4 days 6 MVA-BN ® 5 50×  4 days 7 MVA-BN ® 51× 3 days 8 MVA-BN ® 5 12.5×   3 days 9 MVA-BN ® 5 50×  3 days 10MVA-BN ® 5 1× 2 days 11 Dryvax ® 5 1× 4 days 12 Dryvax ® 5 12.5×   4days 14 Dryvax ® 5 50×  4 days 15 Elstree 5 1× 4 days 16 Elstree 512.5×   4 days 17 Elstree 5 50×  4 days

Justification of Group Size

The primary endpoint of the study was efficacy, determined by the levelof protection at day 4 or 5 following intranasal challenge with VV-WR.Based on previous preclinical experience, it was assumed that followingchallenge at least 95% of the vaccinated group is protected, whereas nomore than 5% of the placebo treated group is protected. Using Fisher'sExact test, a group size of 5 versus 5 is sufficient to demonstrate asignificant difference at the two-sided significance level α=0.05 withpower greater than 80%.

Administration of Test Articles for Vaccination

Vaccinations were performed in a microbiological safety cabinet (SW 100040/class II, Holten Lamin Air). Mice were vaccinated with 200 μl ofMVA-BN® (1×10⁸ TCID₅₀/ml) or Saline control (200 μl PBS) via thesubcutaneous route in the skin wrinkle of the hind leg using a 1 ml 29 Gtuberculin insulin syringe (Terumo). Mice receiving Elstree and Dryvax®were anaesthetized before the scarification of the tail: a fresh mixturecontaining 75 mg Ketamine, 5 mg Xylazine and water was prepared, and 80μl of the anaesthetic was administered via the subcutaneous route usinga 1 ml 27 G insulin syringe. All mice belonging to the same cage wereanaesthetized before administering the vaccine. Subsequently, 2.5 μl or8 μl of Elstree or Dryvax®, respectively, was applied via tailscarification.

Lung Challenge Model

The test article (i.e. VV-WR) was administered via the intranasal routein anaesthetised mice in a microbiological safety cabinet (SW 100040/class II, Holten Lamin Air).

A fresh mixture of 75 mg Ketamine, 5 mg Xylazine in water was preparedas anaesthetic. 80 μl of the anaesthetic was administered via thesubcutaneous route using a 1 ml 29 G insulin syringe. All mice belongingto the same cage were anaesthetized before administering the VV-WR testarticle.

Intranasal challenge was performed in a microbiological safety cabinet(SW 1000 40/class II, Holten Lamin Air). The challenge virus workingdilution was removed from the ice and mixed by gently vortexing for afew seconds. 50 μl of the diluted VV-WR test article was measured usinga 100 μl pipette. Each anaesthetized mouse was held by the skin/fur onthe back of the neck and the body was supported in the palm of the samehand. The test item was slowly added into a single nostril of eachmouse. Each mouse was held as described above until the gasping ceased.

Prior to challenge (day 0), and daily following challenge, animals wereobserved to monitor any signs of ill health. Body weight was measuredprior to challenge (day 0), and daily post challenge, until the day ofnecropsy to monitor the development of disease.

The saline group that received 50× the MLD₅₀ of VV-WR exceeded the bodyweight cut off set by the Dyreforsøgstilsyn and was sacrificed on day 4post challenge. The saline groups that received 1× or 12.5× the MLD₅₀ ofVV-WR exceeded the body weight cut off set by the Dyreforsøgstilsyn andwere sacrificed on day 5 post challenge. Vaccinated animals challengedwith 1× the MLD₅₀ of VV-WR were sacrificed on day 5 post challenge,whereas MVA-BN®-vaccinated animals challenged with 12.5× or 50× theMLD₅₀ of VV-WR were sacrificed latest on day 8 post challenge.

Lungs were removed and the total amount of virus in the lungs wasdetermined using a standard plaque assay on Vero cells. Animals wereconsidered completely protected when lung titers were below 5×10³ pfu,the lowest titer detectable using our method of virus titration on Verocells.

4. RESULTS AND DISCUSSION Changes in Body Weight

The effect of vaccination with different smallpox vaccines on the bodyweight, following a challenge 4 days later (i.e., 4 days aftervaccination) with 1× the MLD₅₀ of vaccinia virus strain Western Reserve(VV-WR) was investigated in some groups of this study. As shown in FIG.1, body weight loss in the group of non-vaccinated (Saline control) micechallenged with 1× the MLD₅₀ of VV-WR was first detectable 3 days afterthe challenge. The body weight continued to drop until sacrifice on day5 to an average of 20.9% below the average initial body weight. Asimilar body weight loss was detected in the groups vaccinated withElstree or Dryvax® 4 days prior to challenge with VV-WR; the averagebody weight on day of sacrifice was either 23.2% or 21.1% below theinitial average body weight in these groups, respectively. In the groupvaccinated with MVA-BN® 2 days prior to challenge with VV-WR, the firstbody weight loss (about 4% from average initial body weight) wasdetected 2 days post challenge. The average body weight continued todrop in this group until day 4 post challenge, with an average bodyweight of 17.6% below the initial one. On day 5 post challenge, theaverage body weight did not continue to drop and was 16.7% below theinitial average body weight. In the group vaccinated with MVA-BN® 3 daysprior to challenge, only a small drop in body weight was detected,starting on day 3 post challenge and being maximally 4.2% below theinitial body weight on day 4 post challenge. In this group, the bodyweight recovered on day 5 to initial values. The group of micevaccinated with MVA-BN® 4 days prior to challenge did not show any bodyweight loss following a challenge with 1× the MLD₅₀ of VV-WR.

In a second set of groups, mice were challenged with 12.5× the MLD₅₀ ofVV-WR and body weight of the mice were again monitored prior tochallenge and then daily post challenge. As shown in FIG. 2, body weightloss in the group of non-vaccinated (Saline control) mice was firstdetectable 2 days after the challenge. The body weight continued to dropuntil sacrifice on day 5 to an average of 23.3% below the averageinitial body weight. Thus, the body weight loss was detectable one dayearlier than in the non-vaccinated group challenged with 1× the MLD₅₀ ofVV-WR, and was more pronounced on the day of sacrifice (see FIG. 1). Abody weight loss similar to that shown by the non-vaccinated mice wasdetected in the groups vaccinated with Elstree or Dryvax® 4 days priorto challenge with VV-WR. In the group vaccinated with MVA-BN® 3 daysprior to challenge with VV-WR, a first small body weight loss (about1.7% from average initial body weight) was detected 2 days postchallenge. The average body weight continued to drop in this group untilday 4 post challenge, with an average body weight of 16.1% below theinitial one. Thereafter, the average body weight started to recover inthis group, and on day 8 post challenge an average body weight that was2.3% below the average initial body weight was detected. In the group ofmice vaccinated with MVA-BN® 4 days prior to challenge, an average bodyweight loss of 10.8% compared to the average initial body weight wasdetected on day 2 post challenge. A maximal average body weight loss of13.8% was detected on day 3 post challenge. Recovery of body weight wasdetected on the subsequent days with a similar average body weightdetected 8 days post challenge than that detected prior to challenge.

In a third set of groups, mice were challenged with 50× the MLD₅₀ ofVV-WR and the body weight of the mice was again monitored prior tochallenge and then daily post challenge. As shown in FIG. 3, a firstbody weight loss in the group of non-vaccinated (Saline control) micewas already detectable 1 day post challenge. The average body weightcontinued to drop until sacrifice on day 4 to 20.1% below the averageinitial body weight. Thus, the body weight loss was detectable 2 days or1 day earlier than that in the non-vaccinated group challenged with 1×or 12.5× the MLD₅₀ of VV-WR, respectively. The body weight loss in thegroups vaccinated with Elstree or Dryvax® started to be detectable 2days post challenge, and by day 4 post challenge the mice in thesegroups revealed an average body weight loss of 20.1% or 19.7% from theinitial body weight, respectively. In the group vaccinated with MVA-BN®3 days prior to challenge with VV-WR, the first body weight loss (about1.6% from average initial body weight) was detected the first day postchallenge. The average body weight continued to drop in this group untilsacrifice on day 4 post challenge, with an average body weight of 24.0%below the initial one. In the group of mice vaccinated with MVA-BN® 4days prior to challenge, a first body weight loss was detectable on thefirst day post challenge, and the average body weight continued to dropto 22.5% below the average initial body weight on day 4 post challenge.Recovery of body weight was detected on subsequent days, and on day 8post challenge an average body weight was detected that was 5.1% belowthe average initial one.

Lung Titers

After death or sacrifice of mice, lungs were removed and the totalamount of virus in this tissue was determined using a standard plaqueassay on Vero cells. Animals were considered completely protected whenlung titers were below log 10 3.69 (5×10³ pfu), the lowest titerdetectable using our method of virus titration on Vero cells.

In a first set of groups, mice challenged with 1× the MLD₅₀ of VV-WRwere compared. As shown in FIG. 4, non-vaccinated mice revealed anaverage virus load of log 10 7.81. Mice vaccinated with Elstree orDryvax® 4 days prior to challenge revealed an average lung virus load oflog 10 7.75 and log 10 6.68. Thus, the Elstree vaccinated mice wereunable, and the Dryvax® vaccinated mice were only to some degree able,to prevent lung viral infection. On the other hand, in the group of micevaccinated with MVA-BN® 4 days prior to challenge, no lung viral titerscould be detected and these mice are thus completely protected fromviral infection following intranasal challenge with 1× the MLD₅₀ ofVV-WR. Shortening the interval between vaccination with MVA-BN® andchallenge with VV-WR from 4 to 3 or 2 days, increased the number ofvirus-positive lungs per group to 1 out of 5, or 4 out of 5, withaverage lung viral titers of log 10 3.77 or log 10 4.68, respectively.

In a second set of groups, mice challenged with 12.5× the MLD₅₀ of VV-WRwere compared. As shown in FIG. 4, non-vaccinated mice revealed anaverage virus load of log 10 8.38. Mice vaccinated with Elstree orDryvax® 4 days prior to challenge, revealed an average lung virus loadof log 10 8.17 and log 10 8.00. Thus, the Elstree and the Dryvax®vaccinated mice were unable to prevent lung viral infection. On theother hand, in the group of mice vaccinated with MVA-BN® 4 or 3 daysprior to challenge, no lung viral titers could be detected and thesemice are thus completely protected from viral infection followingintranasal challenge with 12.5× the MLD₅₀ of VV-WR.

In a third set of groups, mice challenged with 50× the MLD₅₀ of VV-WRwere compared. As shown in FIG. 4, non-vaccinated mice revealed anaverage virus load of log 10 8.59. Mice vaccinated with Elstree orDryvax® 4 days prior to challenge revealed an average lung virus load oflog 10 8.49 and log 10 8.25. Thus, the Elstree and the Dryvax®vaccinated mice were unable to prevent lung viral infection. On theother hand, in the group of mice vaccinated with MVA-BN®, no lung viraltiters was detected when vaccination was administered 4 days prior tochallenge. Consequently, these mice are protected from viral infectionfollowing intranasal challenge with 50× the MLD₅₀ of VV-WR. In the groupof mice in which a 3 day interval between MVA-BN® vaccination andchallenge with VV-WR was applied an average lung viral load of log 107.63 was determined. Thus, this group is only to some degree protectedfrom viral infection.

5. CONCLUSIONS

In the present study, described as Example 1, recovery from body weightloss as well as viral lung titers have been determined to indicate“protection” from a lethal intranasal challenge with VV-WR.

Control animals challenged with 1×, 12.5×, or 50× the MLD₅₀ of VV-WRrevealed a continuous loss of body weight and had a high viral load inthe lungs post-mortem (the higher the challenge dose, the higher theviral load detected in the lungs). Thus, these mice were unable tocontrol the infection.

The smallpox vaccine candidate IMVAMUNE™ (MVA-BN®) was able to protectagainst an intranasal challenge with up to 50× the MLD₅₀ of VV-WR. Thisprotection was associated with recovery of body weight after initialbody weight loss and was also associated with lack of virus in thelungs. The higher the challenge dose of VV-WR, the longer the postchallenge period required for body weight recovery in the micevaccinated 4 days prior to the challenge with MVA-BN®. Indeed, whenchallenged with 12.5× the MLD₅₀ of VV-WR body weight recovery wasdetected on day 4 post challenge, whereas body weight recovery wasdetected on day 5 post challenge when mice have been challenged with 50×the MLD₅₀ of VV-WR. In addition, this study clearly revealed that thetime interval between vaccination of mice with MVA-BN® and challengewith 1× the MLD₅₀ of VV-WR can be reduced to 2 days, a time intervalthat is sufficient to enable stabilization of body weight and to obtainreduced lung viral titers following challenge of mice with 1× the MLD₅₀of VV-WR. Furthermore, increasing the challenge doses resulted in anextension in the time interval between vaccination with MVA-BN® andchallenge that is required to obtain protection from the lethalchallenge. Accordingly, in case of challenge with 50× the MLD₅₀ of VV-WRa 3 day interval was not sufficient, whereas following challenge with12.5× the MLD₅₀ of VV-WR a 3 day interval between vaccination andchallenge was sufficient to obtain protection.

In contrast to MVA-BN®, the first and second generation smallpoxvaccines Dryvax® and Elstree, respectively, were unable to protectagainst an intranasal challenge with up to 50× the MLD₅₀ of VV-WR whenadministered 4 days prior to the challenge. The reason for thisdifference might be due to the different routes of administration:MVA-BN® was administered to mice (and is administered in clinical trialsto humans) subcutaneously, whereas Elstree and Dryvax® was administeredto mice (and is administered in clinical trials to humans) viascarification.

In summary, the study described in Example 1 clearly demonstrates thesuperiority of MVA-BN® over Elstree and Dryvax® with regard to onset ofimmune protection.

Example 2 Onset of Protection in MVA-BN®, Elstree, or Dryvax® VaccinatedMice Challenged with 1×, 12.5×, or 50× the MLD₅₀ of Vaccinia VirusVV-Western Reserve (VV-WR) on Days 2 Through 14 Post-Vaccination:Assessment by Body Weight, Lung Titers, and Specific-IgG Titers

As described below, Example 2 summarises a series of studies that haveinvestigated the onset of protection afforded by various smallpoxvaccines, including MVA-BN®, using the VV challenge model in mice. asassessed by measurements of vaccinia-specific IgG titers, in addition tobody weight and lung titers.

1. VIRUSES AND CONTROLS

All viruses and controls were as described above for Example 1.

2. METHODS AND EXPERIMENTAL DESIGN

The Test System was as described above in Example 1.

Vaccination and Challenge Schedule

As illustrated in Table 2, mice (n=5/group) were challenged with either1×, 12.5× or 50× the MLD₅₀ of VV-WR either 7 or 14 days after a singlesubcutaneous (s.c.) administration of either Saline (s.c.) or MVA-BN®.As a comparison to other traditional smallpox vaccines, other groupswere vaccinated with either Dryvax® or Elstree-BN by tail scarification.Control animals receiving VV-WR challenge were sacrificed 4 or 5 daysafter challenge when the mean body weight loss exceeded 20% from initialbody weight, or when the animals suffered from dyspnea.

TABLE 2 Dose and challenge regimen of mice used to investigate the onsetof MVA-BN ® protection. VV-WR Timing of VV-WR Challenge challenge (DaysDose Dose post-Vaccination) Treatment (TCID₅₀) (MLD₅₀) 7 14 Saline 0 1×X X 12.5×   50×  X X MVA-BN ®   1 × 10⁸ 1× X X 12.5×   50×  X X Dryvax ®2.5 × 10⁵ 1× X X 12.5×   50×  X X Elstree-BN 2.5 × 10⁵ 1× X X 12.5×  50×  X X

Justification of Group Size

Based on previous preclinical experience it was assumed that followingchallenge at least 95% of the vaccinated group was protected whereas nomore than 5% of the Saline treated group were protected. Based onFisher's Exact test a group size of 5 versus 5 is sufficient todemonstrate a significant difference at the two-sided significance levelα=0.05 with power greater than 80%.

In-Life Evaluations and Necropsy Preparations

Clinical Signs

Animals were monitored daily for signs of illness; each animal wasrecorded (at the same time each day) as healthy, sick or dead prior toVV-WR challenge (Day 0) and daily following challenge. Body weight wasmeasured prior to VV-WR challenge (Day 0) and daily post challenge,until the day of necropsy to monitor for development of disease.

Blood Collection

Blood (100-150 μl) from the individual mice was collected prior to thefirst vaccine or saline administration and at varying intervalsthroughout the respective study prior to and post-challenge with VV-WR.Respective study sera sample time points are indicated in the resultssection. The blood was collected, and separated using Microtainer™ serumseparation tubes according to the manufacturer's instructions (BectonDickinson). The tubes containing sera from each mouse were labelled andstored below −15° C. until required for ELISA analysis to determinevaccinia specific IgG titers.

Lung Preparation

Lungs were harvested at the end of the observation period post-challengeand placed into 5 ml of DMEM tissue culture medium (Invitrogen)supplemented with 100 U penicillin/100 μg/ml streptomycin (Sigma) and 2%foetal bovine serum, FBS (PAA). The tubes containing lungs from eachmouse were labelled using appropriate computer printed adhesive labelsand stored below −15° C. until required for use in a Vero cell plaqueassay to determine VV-WR viral lung titer.

Vaccinia-Specific ELISA

Vaccinia-specific IgG ELISA titers were determined from serum samples asdescribed in SOP/PRE/005. The antibody titers (log 10) were calculatedby linear regression (OD on the y-axis and log of the sera dilution onthe x-axis) and defined as the serum dilution that resulted in anoptical density (OD 492 nm) of 0.3. The regression analysis wasperformed using the Magread Macro software as described in SOP/IMM/028.Assay acceptance criteria of OD<0.20 and OD>1.0 have been defined forthe negative and positive samples respectively. The mean antibody titerswere illustrated as Geometric Mean Titer (GMT), together with the SEM.

Titration of VV-WR from Lung Samples on Vero Cells

VV-WR viral titers from the prepared lungs were determined by virustitration on Vero cells as described in SOP/PRE/001. Plaque numbers werecounted using photographic images of the plates and a computerisedsystem (Zeiss Imaging System). The resulting plaque counting raw datawere automatically inserted into an Excel spread sheet that was thenused to calculate viral titer as Log 10 PFU (plaque forming units). Forsummarizing the generated data, the calculated viral titers weremanually inserted into a further (Excel) template. For assay acceptance,the positive control sample had to generate plaque numbers ≦150/well inthe 6400× dilution. The detection limit of the assay was Log 10 3.69.

Data Handling

Microsoft Excel 2000 was used to generate templates for datadocumentation. Appropriate raw data was entered manually into theprinted templates and then attached to laboratory notebooks. Manuallyentered data was then transferred into Excel for analysis (e.g.calculation of average values, SEMs and GMTs).

Data Evaluation

Body weight (in grams) was monitored prior to challenge (Day 0) anddaily post challenge. Changes in body weight for the post-challengeperiod were calculated in % using Microsoft Excel for the individualmice. The average change for each group +/− the standard error wascalculated for each time point post challenge. Lung VV-WR and seravaccinia IgG titers (as log₁₀ titers) were expressed as average +/−standard error per test group. Body weight and lung titer data sets wereillustrated graphically using Microsoft Excel.

Statistical analysis was performed using StatXact 6.1 from CytelSoftware and JMP 6.0.0 from SAS. A statistical significance is definedas p<0.05. For the non-parametric multiple comparison tests, a simpleBonferroni correction was made (significance was claimed if p<0.05/Nwhere N was the number of comparisons).

3. RESULTS AND DISCUSSION Vaccinia-Specific Antibody Induction

Although previous studies, including Example 1, have shown that micevaccinated with MVA-BN® generate an equivalent immune response totraditional smallpox vaccines, time course studies have revealed thatthe immune response is induced faster following MVA-BN®, compared to thetraditional vaccines. However, as has previously been reported, there isa relatively high failure rate (20-30%) of administering traditionalsmallpox vaccines by tail scarification in mice. In contrast, there is a100% sera-conversion by ELISA in the mice vaccinated with MVA-BN® at theoptimal dose (1×108 TCID50). Therefore in an initial analysis, theantibody responses induced by the various smallpox vaccines werecompared for up to 22 days post vaccination, and to overcome the problemof vaccine take failures, 5 animals vaccinated with MVA-BN® werecompared (at certain time points) to groups sizes of 25 and 21 forElstree-BN and Dryvax® respectively.

As illustrated in FIG. 5, following the vaccination with MVA-BN®, adetectable antibody titer could be measured in the majority of theanimals (4 out of 5 mice) by day 8 post vaccination, with a GMT of 94.By day 12 there was a 100% sera-conversion and the GMT titer increasedto 548 that continued to rise steadily until day 22 post vaccination(GMT 912). In contrast, no mice vaccinated with either Dryvax® orElstree-BN had sera-converted by day 8 post vaccinated and only moderateantibody responses could be detected in a minority of animals at day 12and 15 post vaccination. It is difficult to completely assess whichanimals failed to induce immune responses, due to the failure inadministering the vaccines, especially at the earlier time points.However, of the 11 animals vaccinated with Elstree-BN that hadsera-converted by day 22, none had sera-converted on day 8; only 3 hadsera converted by day 12, which rose to a total of 7 by day 15 (anadditional 4 mice) and the remaining 4 animals only sera-converted onday 22 post vaccination. Similarly, of the 7 animals vaccinated withDryvax® that had sera-converted by day 22, none had sera-converted onday 8; only 2 had sera converted by day 12, which rose to a total of 5by day 15 (an additional 3 mice) and the remaining 2 animals onlysera-converted on day 22 post vaccination. Thus, seroconversions areinduced within 8-10 days following vaccination with MVA-BN®, whiletraditional vaccines take 12-22 days to induce a similar response inmice.

Challenge of Animals within Two Weeks Post-Vaccination

The experiment described in Example 1 (see above) demonstrated thesuperiority of MVA-BN® over traditional vaccines Elstree and Dryvax® inaffording protection from viral challenges given at days 2, 3, or 4post-vaccination. To further identify the putative time followingvaccination when the traditional smallpox vaccines would induceprotection in the same manner as MVA-BN® vaccinated animals, additionalstudies were performed whereby vaccinated animals were challenged witheither a low (1×MLD₅₀) or standard (50×MLD₅₀) challenge dose at 7 or 14days post vaccination. As expected, the placebo treated groups had amean body weight loss of −24% 5 days post challenge (1×MLD₅₀), with amean VV titer in the lung of log 10 7.62 pfu (FIG. 6A). Both Elstree-BNand Dryvax® induced a partial protection after 7 days when challengedwith the low dose VV-WR, with a 20% (1/5 animals) and 60% (3/5) efficacyrespectively (FIG. 6A). Indeed, 1 and 2 of the Elstree-BN and Dryvax®animals had to be sacrificed on day 4 post challenge, due to a >20%weight loss and in the Dryvax® group these animals represented the majorreason for the mean weight loss on day 4 post challenge (FIG. 6A). By 14days post vaccination, both Elstree-BN and Dryvax® had induced a goodprotection, as both groups of animals lost a minimal amount of mean bodyweight, although one animal in each group did have a positive titer inthe lungs. (Elstree-BN, log 10 7.56 pfu and Dryvax®, log 10 3.70 pfu;FIG. 6A).

As illustrated in FIG. 6C, the group of placebo treated animalschallenged (50×MLD₅₀) 14 days post vaccination had a mean body weightloss of 21% by day 4 post challenge at which time point they weresacrificed due to the ethical constraints (weight loss not to exceed20%). At this time point the placebo animals recorded a mean VV-WR titerof log 10 8.49 pfu in their lungs (FIG. 6D). In contrast, all theanimals vaccinated once with MVA-BN® were fully protected 7 or 14 dayspost vaccination, as these animals had completely cleared the challengeVV from their lungs 5 days post challenge (FIG. 6B and FIG. 6D).Moreover, all MVA-BN® vaccinated animals experienced a weight loss (mean−18.38 to −21.23%) 3 days following the challenge that appeared to beindependent of when they had been vaccinated (i.e. 7 or 14 days beforechallenge), although all animals showed signs of recovery by the end ofthe post challenge observation period (FIG. 6). The animals vaccinatedwith either Elstree-BN or Dryvax® were completely not protected whenchallenged 7 days later. Indeed, all vaccinated animals failed to lastthe full observation period and like the placebo treated animals had tobe sacrificed 4 days post challenge, due to mean weight losses of−20.30% and −22.69% for the Elstree-BN and Dryvax® vaccinated animalsrespectively. These animals had clearly succumbed to the VV-WRchallenge, as mean viral titers of log 10 8.37 pfu (Elstree-BN) and log10 7.38 pfu (Dryvax®) were recovered from their lungs 4 days postchallenge (FIG. 6). However, both traditional smallpox vaccines inducedalmost a complete protection 14 days following vaccination, as 4/5animals were protected in both the Elstree-BN and Dryvax® treatedgroups, as judged by the clearance of the challenge virus from theirlungs 5 days post challenge (FIG. 6). This partial protection was alsoreflected in the recorded body weight losses, with maximal mean weightlosses on day 3 post challenge of −12.19% and −13.49% in the Elstree-BNand Dryvax® treated groups respectively (FIG. 6).

MVA-Specific Antibody Titer and Lung VV-WR Titer

Antibodies are not detected in MVA-BN®-vaccinated mice by ELISA untilday 7 to day 10 post vaccination, and even later for mice vaccinatedwith traditional smallpox vaccines. Therefore, in an attempt to betterunderstand the protective mechanism observed, particularly in theMVA-BN® treated animals, blood was analyzed after the challenge periodfor total IgG titers specific against vaccinia by ELISA.

No antibody titers could be detected in the placebo treated animals postchallenge, clearly indicating that challenge with VV-WR at any of thedoses investigated, did not raise a detectable immune response to VV(Table 3). In the animals vaccinated with MVA-BN® and challenged 3 dayslater with 50×MLD₅₀VV-WR, no antibodies could be detected by ELISA andas reported above, all animals were not protected with a mean VV titerin the lungs that was equivalent to placebo treated animals (Table 3).However, 4 days after vaccination with MVA-BN® all animals had adetectable antibody response, with a GMT of 597 post challenge and allanimals were fully protected from the same lethal challenge of VV-WR(Table 3).

While all MVA-BN® vaccinated animals were fully protected from 4 dayspost treatment, interestingly the GMT increased post challenge thelonger between vaccination and when the challenge took place; with a GMTof 705 and 1516 after challenge on day 7 and 14 respectively (Table 3).Similarly, 7 days after vaccination with either Elstree-BN or Dryvax®the animals had no detectable antibodies post challenge (50×MLD₅₀) andall animals succumbed to the challenge with high VV titers in the lungs(Table 3). Two weeks post vaccination with Elstree-BN, only one animalhad a VV titer in the lung (log 10 8.52 pfu) and this is the only animalthat had no detectable antibodies following challenge. The remaining 4protected animals all had detectable antibodies following challenge,with a GMT of 282. Similarly, the 4 animals vaccinated with Dryvax® thatwere protected when challenged (50×MLD₅₀) 14 days following vaccinationalso all had a detectable antibody response post challenge. Again theone non-protected animal had a lung titer of log 10 7.87 and had nodetectable antibodies following challenge. Interestingly however, allthe MVA-BN® vaccinated animal that were challenged (1×MLD₅₀) 2 days posttreatment had detectable antibodies (GMT of 106) following challenge andwere un-protected with a mean log 10 titer of 4.68. However, even inthis case the viral load in the lungs was lower than expected,indicating that the MVA-BN® had an effect even after two days postvaccination. Indeed, there was a significant (p<5×10−9) negativecorrelation between the induction of antibodies post challenge to theVV-WR titers in the lungs when a Cohen Kappa, test of agreement wasperformed on all values (including the 2 days post MVA-BN® group).

TABLE 3 Immunogenicty and efficacy of MVA-BN ®, Elstree-BN and Dryvax ®vaccinated mice challenged with VV WR (50 × MLD₅₀). Day of Total IgGLung viral Vaccination Challenge titer post load at Dose (post challengesacrifice Treatment^(a) (TCID₅₀) vaccination) (GMT)^(b) (log10)^(c)Saline — 4 1 8.59 +/− 0.14 14 1 8.49 +/− 0.08 MVA-BN ®   1 × 10⁸ 3 17.63 +/− 0.08^(e)** 4 597** 1.00 +/− 0^(d)** 7 705** 1.00 +/− 0** 141516**  1.00 +/− 0** Elstree-BN 2.5 × 10⁵ 4 1 8.49 +/− 0.03 7 1 8.37 +/−0.08 14 282*  2.50 +/− 1.50* Dryvax ® 2.5 × 10⁵ 4 1 8.25 +/− 0.17 7 16.10 +/− 1.30** 14 84* 2.85 +/− 1.34** Total IgG antibody titers areexpressed as GMT. Lung titers are expressed as log10 +/− the standarddeviation from the mean. Statistical significance from the salinecontrol is indicated by * and ** for p < 0.05 and 0.01 respectively.^(a)Mice (n = 5) were immunized with the vaccines below and challengedvia the intranasal route on days 3, 4, 7 or 14 post vaccination with 2 ×10⁶ TCID₅₀ (50 × MLD₅₀). ^(b)Total IgG titers of 1 were assigned whenthe absorbance at a 1:50 starting dilution was below 0.3. ^(c)Controlmice were sacrificed on day 4 post challenge. Vaccinated mice weresacrificed on day 4 or 5 post challenge. Lung viral load determined byplaque assay. ^(d)Mice were sacrificed on day 8 post challenge. ^(e)Lungtiters were statistically different from MVA-BN vaccinated groups withno virus detected in the lungs (p < 0.01)

4. CONCLUSIONS

The studies described above have demonstrated that a single vaccinationwith MVA-BN® induces a detectable antibody response within the firstweek in the majority of animals, whereas traditional vaccines, based onreplicating VV, actually take longer (up to 22 days) to induce theequivalent responses in mice, in non-human primates (Stittelaar et. al.2005, J. Virol. 2005 79:7845-51), and in an in-house Phase I clinicalstudy. The results described in the Examples confirmed that MVA-BN®induced sera-conversion by 7 to 14 days post vaccination, whereastraditional smallpox vaccines peak response was 22 days aftervaccination. The induction of immunity in animals following vaccinationwith Dryvax® or Elstree-BN parallels the induction of the vaccine take,as sera-conversion only begins to become noticeable 12 days postvaccination, after the formation of the pustule following scarification;also, the majority of the animals require between 14 to 22 days tosera-convert, after the formation of the scab. Indeed, this also appearsto be the case for people vaccinated with Dryvax®, as others havereported that the peak antibody response was 22 days post vaccination ina clinical setting (Frey et al., 2002, N Engl J Med 346:1275-1280).

It could be argued that this is an inappropriate comparison as MVA-BN®is given at a 400× higher dose than the traditional smallpox vaccinesElstree and Dryvax®. However, these studies were designed to compare thevaccine regimes recommended for people (and shown optimal for mice),both in terms of dose and route of administration. While higher doses ofDryvax® could change the dynamics of the immune response, this wouldonly be feasible by changing the route of administration (s.c. orintramuscular), something which would raise severe safety concerns, evenfor healthy people. Of note, by 22 days after vaccination, the immuneresponses induced by both MVA-BN® and Dryvax® are equivalent in mice,non-human primates (Stittelaar et. al. 2005, J. Virol. 2005 79:7845-51)and people. This has also been shown for other MVA strains in comparisonto Dryvax® in animal models, including non-human primates (Wyatt et al.,2004, PNAS. 101: 4590-5; Earl et al., 2004, Nature. 428:182-5),suggesting that MVA is not more immunogenic than traditional smallpoxvaccines. Therefore, and without being bound by a specific theory, thesignificantly faster induction of immunity afforded by MVA-BN® comparedto Dryvax®, appears to be associated with the dose, the route ofadministration, or a combination of both.

Indeed, because MVA-BN® fails to replicate, this allows a much higherdose to be administered in one injection. Again without being bound by aspecific theory, this allows for a rapid induction of B and T cells,because following a s.c. injection MVA-BN® will travel directly to thedraining lymph nodes, the sites of specific immune induction, allowingthe direct stimulation of the immune system. In contrast, traditionalsmallpox vaccines rely on administering a small quantity (dose) of thevirus to the skin, which subsequently needs to replicate (leading to theformation of a pustule) in order to stimulate the immune system toinduce the same immune response as MVA-BN®; a process that takes longerthan a bolus injection of MVA.

This lag in the ability of Dryvax® and Elstree-BN to efficientlystimulate the immune response (in comparison to MVA-BN®), was elegantlydemonstrated by the inability of these vaccines to induce protectionuntil 2 weeks post vaccination, whereas MVA-BN® could protect animalswithin 4 days at the same high challenge dose (50×MLD₅₀). Even at lowerchallenge doses (1× & 12.5×MLD₅₀), the traditional smallpox vaccinesfailed to induce any protection within the first week post vaccination,while MVA-BN® could protect as early as 3 days after vaccination. Evenfollowing just 2 days, there were signs that vaccination with MVA-BN®had been beneficial following the low dose challenge (1×MLD₅₀) with VV,as there was a reduction in the challenge titers in the lungs comparedto placebo treated animals and ⅕ MVA-BN® vaccinated animals wasprotected.

While antibodies could not be detected prior to a challenge, especiallyat the earlier time points for the MVA-BN® vaccinated animals (<7 days),these protected animals had sera converted after the challenge. Thus, itis possible that the induction of a specific immunity, rather than theinduction of innate (non-specific) mechanisms (e.g. induction of type Iinterferon, NK cells etc), is a mechanism of protection. Indeed, therewas a highly significant negative correlation between VV titers in thelungs following challenge (non-protection) and sera-conversion by ELISA,even for the animals vaccinated with Dryvax®. While only totalantibodies were measured in these studies, it has been shown thatprotection from lethal challenges with VV in mice requires bothantibodies and T cells (Wyatt et al., 2004). Given that the ELISA is amore sensitive assay compared to cell based assays for neutralizingantibodies or T cell responses, and that total antibodies correlate toneutralizing antibodies in animals and people vaccinated with MVA-BN®(Stittelaar et. al. 2005, J. Virol. 2005 79:7845-51), it appears thatsera-conversion by ELISA represents an extremely sensitive assay that ispredictive of the induction of a specific immune response. Therefore,while total antibodies measured by ELISA are no more a single correlateof protection, they represent a useful immune parameter that predictsprotection following vaccination with either MVA-BN® or traditionalsmallpox vaccines like Dryvax®.

The data indicate that a factor in the onset of protection is the timerequired to efficiently prime the immune response, such that it hassufficient time to mature and clear the challenge VV. This was drawnfrom the relationship between the times required for protectionfollowing vaccination with MVA-BN®, to the challenge dose of VV-WR.MVA-BN® vaccinated animals could protect by day 4 post challenge, butnot by day 3 at the same challenge dose (50×MLD₅₀), whereas at the lowerchallenge doses (1× & 12×MLD₅₀), MVA-BN® could afford protection a dayearlier (day 3). The unprotected animals failed to mount an immuneresponse and had not sera-converted after the challenge. Also, the onlyanimals that did sera-convert after the challenge, but failed to inducea full protection 5 days following the challenge, were the animalsvaccinated with MVA-BN® and challenged 2 days later. However, given thatthe mean lung titers were lower than expected from the placebo controls,the results indicate that MVA-BN® did have an effect, but maybe thetiming between vaccination and challenge was insufficient to induce anefficient priming of B and T cells to be able to respond and clear thechallenge VV within 5 days.

Although post exposure animal models are difficult to develop to mimicthe natural situation of variola in people, investigating the onset ofprotection and comparing this to the gold standard Dryvax®, providesprobably the best model to investigate efficacy in a post exposureevent. Using this model, Examples 1 and 2 have shown that MVA-BN®(IMVAMUNE®) is superior at inducing a protective immune response inanimal models compared to Dryvax®.

1. A method for inducing a immune response against an infectious agentin an animal, comprising administering to the animal an immunogeniccomposition comprising an MVA at 7 to 2 days prior to infection with theinfectious agent.
 2. The method of claim 1, wherein the infectious agentis a replication competent poxvirus.
 3. The method according to claim 1,wherein the animal is a human.
 4. The method according to claim 3,wherein the MVA is administered in a dose of 10⁵ to 5×10⁸ TCID₅₀/ml. 5.The method according to claim 1, wherein the MVA is administeredintravenously, intramuscularly, or subcutaneously.
 6. The methodaccording to claim 1, wherein the MVA is MVA-BN.
 7. The method accordingto claim 1, wherein the MVA is a recombinant MVA.
 8. The methodaccording to claim 7, wherein the MVA comprises at least oneheterologous nucleic acid sequence coding for at least one antigenicepitope.
 9. The method according to claim 8, wherein the antigenicepitope is an antigenic epitope of the infectious agent.
 10. The methodaccording to claim 9, wherein the infectious agent is selected fromviruses, fungi, pathogenic unicellular eukaryotic and prokaryoticorganisms, and parasitic organisms. 11-18. (canceled)
 19. A kitcomprising an immunogenic composition comprising an MVA and instructionsto administer the immunogenic composition to an animal at a time pointbetween 7 and 2 days prior to exposure to an infectious agent.
 20. Thekit of claim 19, wherein the MVA is MVA-BN at dose of 10⁵ to 5×10⁸TCID₅₀/ml.
 21. The kit of claim 19, wherein the infectious agent issmallpox.
 22. The kit of claim 19, wherein the infectious agent isbacillus anthracis.
 23. The kit of claim 19, wherein the animal is ahuman.
 24. The kit of claim 19, wherein the MVA is a recombinant MVA.25. The kit of claim 19, comprising instructions to deliver theimmunogenic composition at a time point between 6 and 2 days prior toexposure to an infectious agent.
 26. The kit of claim 19, comprisinginstructions to deliver the immunogenic composition at a time pointbetween 5 and 2 days prior to exposure to an infectious agent.
 27. Thekit of claim 19, comprising instructions to deliver the immunogeniccomposition at a time point between 4 and 2 days prior to exposure to aninfectious agent.
 28. The kit of claim 19, comprising instructions todeliver the immunogenic composition at a time point between 3 and 2 daysprior to exposure to an infectious agent.